Immunology 1990 69 1-7
Analysis of immune responses in the sheep to synthetic peptides of foot-and-mouth disease virus using ovine polyclonal and monoclonal antibodies J. N. FLYNN,* G. D. HARKISS,* T. DOELt & R. DiMARCHIt *Department of Veterinary Pathology, University of Edinburgh, Edinburgh, tAgricultural and Food Research Council, Institute of Animal Health, Pirbright, Surrey and tEli Lilly Research Laboratories, Indianapolis, U.S.A.
Acceptedfor publication 27 September 1989
SUMMARY A 40-residue peptide incorporating residues 200-213 and 141-158 of foot-and-mouth disease virus VP1 capsid protein strain 01 Kaufbeuren was injected uncoupled into sheep, and the immune responses analysed. Direct-binding and inhibition experiments showed that the polyclonal antibody response was directed mainly against epitopes unique to the 40-residue peptide but absent from the constituent peptides containing residues 200-213 or 141-158, respectively. Further confirmation of the presence of unique epitopes on the 40-residue peptide was obtained from similar experiments performed with sheep monoclonal antibodies generated through the use of an aminopterin-sensitive sheep/mouse heterohybridoma cell line as a fusion partner. The sheep polyclonal antisera to the 40residue peptide had high neutralization titres and were fully active in a mouse protection assay, whereas none of the sheep monoclonal antibodies conferred protection. The results suggest that the conformation of the 40-residue peptide is important for its ability to induce neutralizing antibodies.
INTRODUCTION The immunodominant epitopes of foot-and-mouth disease virus (FMDV) are known to reside on VP1, one of the four capsid proteins of the virus (Wild, Burroughs & Brown, 1969; Bachrach et al., 1975; Meloen, Rowlands & Brown, 1979; Strohmaier, Franze & Adam, 1982). It has been demonstrated further that the major epitopes involve VP1 residues in the region 141-160 and to a lesser extent 200-213 (Pfaffet al., 1982; Bittle et al., 1982), and that synthetic peptide analogues of these regions can elicit virus neutralizing antibodies in experimental animals (Bittle et al., 1982; Francis et al., 1985; Francis et al., 1988). However, it has proven difficult to induce protective immune responses in target veterinary species using these peptides. As a consequence, attempts have been made to identify more precisely the epitopes associated with these regions of the virus (Rowlands et al., 1983; Parry et al., 1985; Geysen, Barteling & Meloen, 1985; Meloen et al., 1987; Xie et al., 1987). In addition, efforts have been made to improve the immunogenicity of the peptide sequences through the generation of fusion proteins (Clarke et al., 1987; Winther et al., 1987) or the introduction of exogenous T-cell epitopes (Francis et al., 1987). In a previous report, DiMarchi et al. (1986) showed that a 40-residue hybrid peptide consisting of the 200-213 sequence
linked via a diproline spacer to the 141-158 sequence was effective in protecting cattle from live viral challenge. Subsequent analysis of variants of the hybrid peptide showed that larger or smaller peptides were less effective at inducing neutralizing antibodies in guinea-pigs (Doel et al., 1988). In the present report, we have analysed immune responses to this 40residue tandem peptide, as well as to peptides representing the two constituent sequences in sheep. We show that the 40-residue peptide not only induces neutralizing antibodies but also, through the use of sheep monoclonal antibodies, that a substantial component of the antibody response is directed against unique epitopes which are not present on the two constituent 21- and 19-residue peptides.
MATERIALS AND METHODS FMD V synthetic peptides The 40-residue and 21-residue peptides described previously by DiMarchi et al. (1986) were used in this study (Table 1). The 21 residue peptide contained the 01 Kaufbeuren (01K) strain FMDV VP1 sequences 141-158, with an additional carboxylterminal proline-cysteine-glycine triplet. The 40-residue peptide contained VP1 sequences 200-213 linked via a diproline serine spacer to residues 141-158, and contained two amino-terminal cysteines and proline-cysteine-glycine residues at the carboxy terminus. A 19-amino acid peptide containing VPl residues 200-213, and a 20-residue control peptide from a Mycobacter-
Correspondence: Dr J. N. Flynn, Dept. of Veterinary Pathology, University of Edinburgh, Edinburgh EH9 IQH, U.K.
1
J. N. Flynn et al.
2
Table 1. Amino acid sequences of FMDV and tuberculin peptides
Peptide designation
Peptide sequence*
FMDV peptides 40-residue 21-residue
CC-200-213-PPS-141-158-PCG 141-158-PCG 19-residue CC-200-213-PPS Tuberculin peptide NGSQMRLIADVGPESATVAKC *
One letter code for amino acids. The numbers
represent residue numbers of the 01K strain FMDV VP1
capsid protein sequence. ium tuberculosis sequence (Kuwabara, 1975) with an added carboxy-terminal cysteine residue were synthesized in the Department of Veterinary Pathology by application of the fluoronylmethoxycarbonyl (Fmoc) peptide chemistry described by Dryland & Sheppard (1986). Amino acids for incorporation into the peptide were supplied as Fmoc pentafluorophenyl esters (MilliGen, Millipore U.K. Ltd, Watford, Herts, U.K.) containing side-chain protecting groups where appropriate. A solidphase peptide synthesizer (Pepsynthesiser II; Cambridge Research Biochemicals Ltd, Cambridge, U.K.) was used throughout. The lyophilized peptides were desalted on a Sephadex G1O gel filtration column (Pharmacia Ltd, Milton Keynes, Bucks, U.K.) and purified by reversed-phase chromatography on a C18 Zorbax ODS column (Du Pont, Wilmington, DE, U.S.A.) using a gradient of acetonitrile (Rathburn Chemicals Ltd, Walkerburn, Scotland; HPLC grade) from 0% to 100% in 0-1% TFA, pH 2, over a period of 30 min. The amino acid content of the peptides synthesized in the Department were checked by amino acid analysis (performed by the Department of Biochemistry, University of Edinburgh). The 19-residue peptide of FMDV was coupled to ovalbumin (OVA; Sigma, Poole, Dorset, U.K.) using glutaraldehyde.
Immunization of sheep Sheep were injected with 200 pg of the synthetic peptides, uncoupled unless otherwise stated, emulsified in Freund's complete adjuvant (FCA) on two occasions spaced 2 weeks apart. In the case of the 19-residue synthetic peptide coupled to OVA, approximately 100 pg of peptide linked to 100 pg ovalbumin were injected each time. Three sheep were immunized with each peptide. Serum samples were taken 2 weeks after the second injection of antigen. Detection of sheep antibodies to the 40-, 21- and 19-residue FMD V peptides The wells of ELISA plates were coated with peptide antigens at 10 pg/ml 15 mm carbonate buffer, pH 9-6, overnight at 4°. Doubling dilutions of sheep serum were prepared in phosphatebuffered saline (PBS) containing 2% bovine serum albumin (BSA). Sheep anti-peptide antibodies were detected directly using peroxidase-labelled donkey anti-sheep immunoglobulin (Scottish Antibody Production Unit, Carluke, Scotland) diluted 1:1000 in PBS/BSA. All incubation periods were for 1 hr at room temperature. The results were expressed as absorbance values at 492 nm. For the detection of sheep monoclonal
antibodies in culture supernatant, the ELISA plates were coated with peptides then monoclonal antibody supernatants added for 1 hr at room temperature. After washing, a murine monoclonal antibody (VPM8) to sheep immunoglobulin light chains, generated in the Department of Veterinary Pathology, was added for I hr at room temperature. The bound murine monoclonal antibodies were detected using a sheep anti-mouse immunoglobulin-peroxidase conjugate generated in the Department of
Veterinary Pathology. Inhibition assays Serum samples were diluted 1/500 in PBS/BSA and incubated with either the 40-residue or 21-residue peptides for I hr at room temperature. The concentration of peptide added ranged from 100 ug/ml to 0 038 pg/ml in doubling dilutions. The peptide/ serum mixtures were added to wells of ELISA plates coated with peptides as described above. Thereafter, standard ELISA procedures were followed. The results were expressed as percentage inhibition, calculated from values obtained with control serum samples without added aqueous peptide. Surgical removal of lymph nodes The right popliteal lymph node was removed under general anaesthesia from a sheep primed with the 40-residue FMDV peptide. Anaesthesia was induced by intravenous injection of 9 mg/kg 'Saffan' (Glaxovet Ltd, Uxbridge, Middlesex, U.K.), and maintained with 3% 'Fluothane' (Coopers A.H. Ltd, Crewe, Cheshire, U.K.) administered in oxygen. Four days prior to excision of the lymph node, it was stimulated antigenically by the injection of 100 pg of the 40-residue peptide in sterile normal saline at three sites intradermally into the drainage area of the node.
Generation of sheep monoclonal antibodies to the 40-residue FMD V peptide Lymph node cells were prepared by dissecting the node free of surrounding fat tissue then cutting the node into small pieces approximately 4 mm diameter. The lymph node tissue was homogenized by gently forcing the tissue through a sterile nylon filter. The cells were washed twice with serum-free RPMI medium and were used immediately. The lymph node cells were fused to an aminopterin-sensitive sheep/mouse heterohybridoma cell line, denoted lC6.3a 6T. 1D7 (Flynn, Harkiss & Hopkins, 1989), at a ratio of two lymphocytes: 1 lC6.3a 6T. 1D7 heterohybridoma cell using polyethylene glycol. Thereafter, the fusion protocol was the same as described previously (Flynn et al., 1989). The sheep monoclonal antibody supernatants were screened undiluted by ELISA, as described earlier. Anti-viral assays Virus neutralization tests were determined in a microtitre test using the porcine cell line IB-RS2 (Golding et al., 1976). The results are expressed as the dilution factor required to neutralize 50% of the viral infectivity using 100 TCIDm units of homologous virus. A mouse protection assay was carried out by injecting 0-1 ml of serum or monoclonal antibody supernatant dilutions subcutaneously into 5 x 5 groups of mice. One hour later, the mice were challenged intraperitoneally with 0-03 ml of dilutions over 4 logs of mouse-adapted 01 FMDV. The logio lethal dosem was calculated on the basis of death or paralysis 7 days after virus challenge. A titre of
Analysis of immune responses in the sheep to synthetic peptides of foot-and-mouth disease virus using ovine polyclonal and monoclonal antibodies J. N. FLYNN,* G. D. HARKISS,* T. DOELt & R. DiMARCHIt *Department of Veterinary Pathology, University of Edinburgh, Edinburgh, tAgricultural and Food Research Council, Institute of Animal Health, Pirbright, Surrey and tEli Lilly Research Laboratories, Indianapolis, U.S.A.
Acceptedfor publication 27 September 1989
SUMMARY A 40-residue peptide incorporating residues 200-213 and 141-158 of foot-and-mouth disease virus VP1 capsid protein strain 01 Kaufbeuren was injected uncoupled into sheep, and the immune responses analysed. Direct-binding and inhibition experiments showed that the polyclonal antibody response was directed mainly against epitopes unique to the 40-residue peptide but absent from the constituent peptides containing residues 200-213 or 141-158, respectively. Further confirmation of the presence of unique epitopes on the 40-residue peptide was obtained from similar experiments performed with sheep monoclonal antibodies generated through the use of an aminopterin-sensitive sheep/mouse heterohybridoma cell line as a fusion partner. The sheep polyclonal antisera to the 40residue peptide had high neutralization titres and were fully active in a mouse protection assay, whereas none of the sheep monoclonal antibodies conferred protection. The results suggest that the conformation of the 40-residue peptide is important for its ability to induce neutralizing antibodies.
INTRODUCTION The immunodominant epitopes of foot-and-mouth disease virus (FMDV) are known to reside on VP1, one of the four capsid proteins of the virus (Wild, Burroughs & Brown, 1969; Bachrach et al., 1975; Meloen, Rowlands & Brown, 1979; Strohmaier, Franze & Adam, 1982). It has been demonstrated further that the major epitopes involve VP1 residues in the region 141-160 and to a lesser extent 200-213 (Pfaffet al., 1982; Bittle et al., 1982), and that synthetic peptide analogues of these regions can elicit virus neutralizing antibodies in experimental animals (Bittle et al., 1982; Francis et al., 1985; Francis et al., 1988). However, it has proven difficult to induce protective immune responses in target veterinary species using these peptides. As a consequence, attempts have been made to identify more precisely the epitopes associated with these regions of the virus (Rowlands et al., 1983; Parry et al., 1985; Geysen, Barteling & Meloen, 1985; Meloen et al., 1987; Xie et al., 1987). In addition, efforts have been made to improve the immunogenicity of the peptide sequences through the generation of fusion proteins (Clarke et al., 1987; Winther et al., 1987) or the introduction of exogenous T-cell epitopes (Francis et al., 1987). In a previous report, DiMarchi et al. (1986) showed that a 40-residue hybrid peptide consisting of the 200-213 sequence
linked via a diproline spacer to the 141-158 sequence was effective in protecting cattle from live viral challenge. Subsequent analysis of variants of the hybrid peptide showed that larger or smaller peptides were less effective at inducing neutralizing antibodies in guinea-pigs (Doel et al., 1988). In the present report, we have analysed immune responses to this 40residue tandem peptide, as well as to peptides representing the two constituent sequences in sheep. We show that the 40-residue peptide not only induces neutralizing antibodies but also, through the use of sheep monoclonal antibodies, that a substantial component of the antibody response is directed against unique epitopes which are not present on the two constituent 21- and 19-residue peptides.
MATERIALS AND METHODS FMD V synthetic peptides The 40-residue and 21-residue peptides described previously by DiMarchi et al. (1986) were used in this study (Table 1). The 21 residue peptide contained the 01 Kaufbeuren (01K) strain FMDV VP1 sequences 141-158, with an additional carboxylterminal proline-cysteine-glycine triplet. The 40-residue peptide contained VP1 sequences 200-213 linked via a diproline serine spacer to residues 141-158, and contained two amino-terminal cysteines and proline-cysteine-glycine residues at the carboxy terminus. A 19-amino acid peptide containing VPl residues 200-213, and a 20-residue control peptide from a Mycobacter-
Correspondence: Dr J. N. Flynn, Dept. of Veterinary Pathology, University of Edinburgh, Edinburgh EH9 IQH, U.K.
1
J. N. Flynn et al.
2
Table 1. Amino acid sequences of FMDV and tuberculin peptides
Peptide designation
Peptide sequence*
FMDV peptides 40-residue 21-residue
CC-200-213-PPS-141-158-PCG 141-158-PCG 19-residue CC-200-213-PPS Tuberculin peptide NGSQMRLIADVGPESATVAKC *
One letter code for amino acids. The numbers
represent residue numbers of the 01K strain FMDV VP1
capsid protein sequence. ium tuberculosis sequence (Kuwabara, 1975) with an added carboxy-terminal cysteine residue were synthesized in the Department of Veterinary Pathology by application of the fluoronylmethoxycarbonyl (Fmoc) peptide chemistry described by Dryland & Sheppard (1986). Amino acids for incorporation into the peptide were supplied as Fmoc pentafluorophenyl esters (MilliGen, Millipore U.K. Ltd, Watford, Herts, U.K.) containing side-chain protecting groups where appropriate. A solidphase peptide synthesizer (Pepsynthesiser II; Cambridge Research Biochemicals Ltd, Cambridge, U.K.) was used throughout. The lyophilized peptides were desalted on a Sephadex G1O gel filtration column (Pharmacia Ltd, Milton Keynes, Bucks, U.K.) and purified by reversed-phase chromatography on a C18 Zorbax ODS column (Du Pont, Wilmington, DE, U.S.A.) using a gradient of acetonitrile (Rathburn Chemicals Ltd, Walkerburn, Scotland; HPLC grade) from 0% to 100% in 0-1% TFA, pH 2, over a period of 30 min. The amino acid content of the peptides synthesized in the Department were checked by amino acid analysis (performed by the Department of Biochemistry, University of Edinburgh). The 19-residue peptide of FMDV was coupled to ovalbumin (OVA; Sigma, Poole, Dorset, U.K.) using glutaraldehyde.
Immunization of sheep Sheep were injected with 200 pg of the synthetic peptides, uncoupled unless otherwise stated, emulsified in Freund's complete adjuvant (FCA) on two occasions spaced 2 weeks apart. In the case of the 19-residue synthetic peptide coupled to OVA, approximately 100 pg of peptide linked to 100 pg ovalbumin were injected each time. Three sheep were immunized with each peptide. Serum samples were taken 2 weeks after the second injection of antigen. Detection of sheep antibodies to the 40-, 21- and 19-residue FMD V peptides The wells of ELISA plates were coated with peptide antigens at 10 pg/ml 15 mm carbonate buffer, pH 9-6, overnight at 4°. Doubling dilutions of sheep serum were prepared in phosphatebuffered saline (PBS) containing 2% bovine serum albumin (BSA). Sheep anti-peptide antibodies were detected directly using peroxidase-labelled donkey anti-sheep immunoglobulin (Scottish Antibody Production Unit, Carluke, Scotland) diluted 1:1000 in PBS/BSA. All incubation periods were for 1 hr at room temperature. The results were expressed as absorbance values at 492 nm. For the detection of sheep monoclonal
antibodies in culture supernatant, the ELISA plates were coated with peptides then monoclonal antibody supernatants added for 1 hr at room temperature. After washing, a murine monoclonal antibody (VPM8) to sheep immunoglobulin light chains, generated in the Department of Veterinary Pathology, was added for I hr at room temperature. The bound murine monoclonal antibodies were detected using a sheep anti-mouse immunoglobulin-peroxidase conjugate generated in the Department of
Veterinary Pathology. Inhibition assays Serum samples were diluted 1/500 in PBS/BSA and incubated with either the 40-residue or 21-residue peptides for I hr at room temperature. The concentration of peptide added ranged from 100 ug/ml to 0 038 pg/ml in doubling dilutions. The peptide/ serum mixtures were added to wells of ELISA plates coated with peptides as described above. Thereafter, standard ELISA procedures were followed. The results were expressed as percentage inhibition, calculated from values obtained with control serum samples without added aqueous peptide. Surgical removal of lymph nodes The right popliteal lymph node was removed under general anaesthesia from a sheep primed with the 40-residue FMDV peptide. Anaesthesia was induced by intravenous injection of 9 mg/kg 'Saffan' (Glaxovet Ltd, Uxbridge, Middlesex, U.K.), and maintained with 3% 'Fluothane' (Coopers A.H. Ltd, Crewe, Cheshire, U.K.) administered in oxygen. Four days prior to excision of the lymph node, it was stimulated antigenically by the injection of 100 pg of the 40-residue peptide in sterile normal saline at three sites intradermally into the drainage area of the node.
Generation of sheep monoclonal antibodies to the 40-residue FMD V peptide Lymph node cells were prepared by dissecting the node free of surrounding fat tissue then cutting the node into small pieces approximately 4 mm diameter. The lymph node tissue was homogenized by gently forcing the tissue through a sterile nylon filter. The cells were washed twice with serum-free RPMI medium and were used immediately. The lymph node cells were fused to an aminopterin-sensitive sheep/mouse heterohybridoma cell line, denoted lC6.3a 6T. 1D7 (Flynn, Harkiss & Hopkins, 1989), at a ratio of two lymphocytes: 1 lC6.3a 6T. 1D7 heterohybridoma cell using polyethylene glycol. Thereafter, the fusion protocol was the same as described previously (Flynn et al., 1989). The sheep monoclonal antibody supernatants were screened undiluted by ELISA, as described earlier. Anti-viral assays Virus neutralization tests were determined in a microtitre test using the porcine cell line IB-RS2 (Golding et al., 1976). The results are expressed as the dilution factor required to neutralize 50% of the viral infectivity using 100 TCIDm units of homologous virus. A mouse protection assay was carried out by injecting 0-1 ml of serum or monoclonal antibody supernatant dilutions subcutaneously into 5 x 5 groups of mice. One hour later, the mice were challenged intraperitoneally with 0-03 ml of dilutions over 4 logs of mouse-adapted 01 FMDV. The logio lethal dosem was calculated on the basis of death or paralysis 7 days after virus challenge. A titre of
